Molecular Mapping Of The Genetic Multiple-allele Male-serile Gene In Chinese Cabbage

As the first step toward cloning the multiple-allele male-sterile gene Ms in Chinesecabbage, we attempted the molecular tagging of this male-sterility locus using the AFLP andSSR techniques. A BC₁ mapping population (244 plants) segregating for male sterility/fertilitywas developed for the tagging using the bulk segregant analysis. A set of AFLP and SSRmarkers were identified to be linked to the Ms gene. After the two AFLP markers which flankthe Ms gene were converted to SCAR markers, the Ms gene was mapped with the SCAR andSSR markers. On the other hand, we preliminarily verified the allelic relationship between theMs and Ms^f genes using SSR technique, with an AB line as the mapping population. All theresults were described as follows:1. According to the segregation ratio for fertility/sterility in the progenies from thetestcrosses between Chiifu and four genotype-identified materials, Chiifu was identified asmsms. Then, one F₁ plant, generated from the cross between Chiifu as the male parent and themale-sterile plant of AB01 as the female parent, was backcrossed with Chiifu, to develop aBC₁ population. In the BC₁ population, 114 were fertile, while 130 were male sterile.2. Using the AFLP+BSA technique, 256 PstI+GNN/MseI+CNN primer combinationswere screened to identify AFLP markers linked to the Ms gene. In the 16 candidate markersobtained in the screening among two parents and two bulks, six were finally verified to beclosely linked to the Ms gene. By using the MAPMAKER/EXP 3.0, the Ms gene was mappedwith the associated AFLP markers. The linkage map covers a total interval of 15 cM. P01 andP04 flank the gene at 2.5 and 3.8 cM, respectively. All the other markers (P10, P12, P07 andP11) were mapped on the same side as P04, at genetic distances of 6.3, 10, 12.5 and 12.5 cM,respectively.3. The nearest two AFLP markers (P01 and P04) which flank the Ms gene were clonedand sequenced. New primers were designed according to the sequence of the two markers.Two SCAR markers named syau_scr01 and syau_scr04 were successfully converted from thecorresponding AFLP markers. In case the two markers were used in combination, thetheoretical correct rate might reach 100% in the marker-assisted selection.4. In order to tag the Ms gene with SSR markers, the SSR-PCR system was optimized forChinese cabbage. The optimized system is as follows: 1×buffer (10 mmol·L^-1 Tris-HCL, 50 mmol·L^-1 KCL), 2 ng·μl^-1 module DNA, 1U Taq DNA polymerase, 0.35 mmol·L^-1 dNTPs,0.4μmol·L^-1 primer, 2 mmol·L^-1 Mg²⁺, in a 20-μl volume. Further, 286 SSR primers whichcover the whole genome of Chinese cabbage were screened between two parents, AB01-1 andChiifu. In the 286 primers, 182 (68%) were identified to be polymorphic. In the furtherverification of these primers on two bulks and 244 individuals, three SSR markers (cnu_m273,cnu_m030, and cnu_m295) were identified to be tightly linked to the Ms gene. All these threemarkers were originated from the A07 linkage group of Chinese cabbage. In order to obtainSSR markers more closely linked to the gene, 14 primers were designed for the SSRs on theseven BAC clones on A07. One more SSR marker was identified linked to the Ms gene.Through genetic linkage analysis, a linkage map was constructed with the SSR and SCARmarkers, covering a total interval of 9 cM. Two SCAR markers, syau_scr01 and syau_scr04,flank the Ms gene at 0.8 and 2.5 cM. All the four associated SSR markers (syau_m13,cnu_m273, cnu_m030, and cnu_m295) were mapped on the same side as syau_scr04, atdistances of 3.3, 6.2, 7.8, and 8.6, respectively. On the basis of the reference genetic linkagemap of the A07 linkage group which was constructed by BrGSP, the Ms gene was located inthe 3,3-cM region near the 26.9-cM position on the A07 linkage group.5. With the AB01 (an AB line of Chinese cabbage) as the mapping population, all the 40SSR primers on the A07 linkage group were screened between the fertile (Ms^f Ms) and sterile(MsMs) bulks. One SSR marker, named pbc_mENA6a, was identified to be linked to therestorer gene Ms^f at a distance of 7.8 cM. In order to verify the allelic relationship between theMs and Ms^f genes, we compared the two SSR linkage maps associating the Ms and Ms^f alleleswith the genetic linkage map of the A07 linkage group. The results indicated that the twogenes were mapped in an interval of 0-26.9 cM. The result is valuable for revealing themolecular mechanism on the target locus, though the allelic relationship between the Ms andMs^f genes could not be verified thoroughly in the present study.